1. Technical Field
The present disclosure is related generally to methods for culturing animal cells in the presence of exogenous lactate.
2. Description of the Related Art
A number of successful methods have been developed for the commercial production of recombinant therapeutic proteins and other products using animal cell culture. The fed-batch approach is among the more popular methods, as it works well for current good manufacturing process (cGMP) operations and provides improved cell culture performance compared to the batch method. It involves minimally invasive operations and allows predictable scale-up. To sustain cell performance over time in such cultures, a strategy of providing nutrients sufficient for high cell growth, viability, and productivity becomes important. For practical reasons, and to avoid the risk of nutrient depletion, the levels of many key nutrients, such as glucose, are often above the minimum required amount. When glucose is above the minimum required amount, it is not metabolized efficiently. As a result, lactic acid is generated and can accumulate to concentrations that inhibit cell growth and recombinant protein formation (Glacken et al., Biotechnol Bioeng. 1988, 32:491-506; Lao and Toth, Biotechnol Prog. 1997, 13:688-691; Omasa et al., Biotechnol Bioeng. 1992, 39:556-564; Ozturk et al., Biotechnol Bioeng. 1992, 39:418-431). Inhibition of cell growth is often partly due to excessive increases in osmolality in pH-controlled bioreactors; this is a particular challenge for fed-batch processes where the extension of culture time can be accompanied by excessive lactic acid accumulation (Chu and Robinson, Curr Opin Biotechnol 12:180-1872001; Hu and Ozturk, Cell Culture Technology for Pharmaceutical and Cell-Based Therapies. Florida: CRC Press 2006; Meier, 2005, “Cell culture scale-up: mixing, mass transfer, and use of appropriate scale-down models”, Biochemical engineering XIV. Harrison Hot Springs, Canada).
To meet this challenge, a number of strategies have been developed to minimize lactic acid production. Simple methods, such as control of the culture at lower pH, have been implemented in manufacturing operations, but are often insufficient. Furthermore, low pH levels may negatively impact the growth, viability, or productivity of the cells, as well as the quality of any product derived from or generated by the cells. More complicated strategies, such as feedback control of glucose at low levels (Altamirano et al., Biotechnol Bioeng. 2001, 76:351-360; Maranga and Goochee, Biotechnol Bioeng. 2006, 94:139-150; Xie and Wang, Cytotechnology. 1994, 15:17-29; Zhou et al., Biotechnol Bioeng. 1995, 46:579-587) may be considered not robust enough for implementation in actual cGMP operations. Low glucose levels may lead to glucose depletion, apoptosis, and premature cell death (Yeo et al., Biotechnol Lett. 2006, 28:1445-1452) or affect product quality by reducing glycosylation (Nyberg, IBM Journal of Research and Development. 2000, 44:770). These considerations make implementation less attractive.
Use of alternative sugars, such as fructose and galactose, and/or other glucose analogs that metabolize more slowly, produces reduced byproducts (Altamirano et al., Biotechnol Bioeng. 2000, 76:351-360; Duval et al., Hybridoma. 1992, 11:311-322) and may also lead to lower growth rates or reduced effectiveness. Metabolic engineering approaches to limiting glycolytic activity (Paredes et al. Cytotechnology. 1999, 30:85-93), disrupt lactate dehydrogenase (Chen et al. Biotechnol Bioeng. 2001, 72:55-61; Jeong et al. Biochem Biophys Res Commun. 2001, 289:1141-1149) or improve flux into the TCA cycle (Irani et al. Biotechnol Bioeng. 1999, 66:238-246) are often time consuming and have the potential for creating unstable cell lines in CHO cell cultures. These methods have improved over the years (Kim and Lee, Appl Microbiol Biotechnol. 2007, 74:152-159; Kim and Lee, Appl Microbiol Biotechnol. 2007, 76:659-665) but have not been significantly implemented in commercial processes. There still exists a need to develop a strategy that restricts lactic acid production in a simple and robust manner.